title: Role of the insertion domain and the zinc-finger motif of Escherichia coli UvrA in damage recognition and ATP hydrolysis.
abstract: UvrA is the initial DNA damage-sensing protein in bacterial nucleotide excision repair. Each protomer of the UvrA dimer contains two ATPase domains, that belong to the family of ATP-binding cassette domains. Three structural domains are inserted in these ATPase domains: the insertion domain (ID) and UvrB binding domain (in ATP domain I) and the zinc-finger motif (in ATP domain II). In this paper we analyze the function of the ID and the zinc finger motif in damage specific binding of Escherichia coli UvrA. We show that the ID is not essential for damage discrimination, but it does stabilize UvrA on the DNA, most likely by forming a clamp around the DNA helix. We present evidence that two conserved arginine residues in the ID contact the phosphate backbone of the DNA, leading to strand separation after the ATPase-driven movement of the ID's. Remarkably, deletion of the ID generated a phenotype in which UV-survival strongly depends on the presence of photolyase, indicating that UvrA and photolyase form a ternary complex on a CPD-lesion. The zinc-finger motif is shown to be important for the transfer of the damage recognition signal to the ATPase of UvrA. In the absence of this domain the coupling between DNA binding and ATP hydrolysis is completely lost. Mutation of the phenylalanine residue in the tip of the zinc-finger domain resulted in a protein in which the ATPase was already triggered when binding to an undamaged site. As the zinc-finger motif is connected to the DNA binding regions on the surface of UvrA, this strongly suggests that damage-specific binding to these regions results in a rearrangement of the zinc-finger motif, which in its turn activates the ATPase. We present a model how damage recognition is transmitted to activate ATP hydrolysis in ATP binding domain I of the protein.
authors.affiliation: Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, The Netherlands.
authors.forename: Koen
authors.initials: K
authors.lastname: Wagner
authors.forename: Geri F
authors.initials: GF
authors.lastname: Moolenaar
authors.forename: Nora
authors.initials: N
authors.lastname: Goosen
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D050296:Microbial Viability / Q000528:radiation effects; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D014466:Ultraviolet Rays / Q000009:adverse effects; D016335:Zinc Fingers
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D000255:Adenosine Triphosphate; D001120:Arginine; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004255:Deoxyribodipyrimidine Photo-Lyase